The long term objective of this application is to understand the molecular mechanisms of the Drosophila innate immune response, a process by which genes encoding antifungal and antibacterial peptides are activated in response to infection by pathogenic microorganisms. The specific aims are to identify and functionally characterize genes that encode essential proteins in the signaling pathways leading to the activation of immune response genes. A number of different approaches will be taken to identify these genes, including bioinformatic searches of the growing Drosophila DNA data base with sequences of genes known to be involved in the human innate immune response, yeast two-hybrid experiments using cDNAs encoding known Drosophila immune response pathway genes, and biochemical purification and microsequencing of immune response proteins followed by cDNA cloning. The proteins encoded by these genes will be functionally characterized using cell transfection and embryo microinjection assays and more conventional genetic approaches. Initially, efforts will be made to characterize CAKalpha, (Cactus Kinase alpha) a putative Drosophila homologue of the human IkappaB kinases IKKalpha and IKKbeta, and CAKgamma, a putative homologue of the human IkappaB kinase complex component IKKgamma. Stable cell lines expressing epitope-tagged versions of these proteins will be established and used to immunoaffinity purify other components of the CAK complex, which will be microsequenced and the information used to isolate and characterize the corresponding cDNA clones. Other components of the immune response signaling pathway, including dTRAF6 and dTRAF4, putative homologs of the human TRAF family of signal transduction factors will be functionally characterized. Because of the high degree of evolutionary conservation between the innate immune responses in humans and Drosophila, these studies should provide significant new insights into the mechanisms involved in anti-microbial responses in humans. In addition, the signaling pathways involved in these processes have been implicated in human inflammatory diseases, such as arthritis. Thus, the information gained in these studies will be of direct relevance to the understanding of human infectious and inflammatory diseases. The ability to exploit Drosophila genetics should provide information that cannot be directly obtained from studies of the human innate immune response.